摘要
目的探讨表皮剥脱毒素A(ETA)对体外培养正常人角质形成细胞表面天疱疮抗原的影响及其作用机制。方法人角质形成细胞复层化表达落叶型天疱疮抗原后与ETA作用,分别用寻常型或落叶型天疱疮抗体进行免疫荧光染色,提取培养细胞总蛋白做免疫印迹;测定ETA作用后细胞上清液中IL-1α,IL-6的浓度及其分解酪蛋白的活性。结果ETA作用前后细胞间落叶型天疱疮抗体着色由强的连续发光转变为弱的断续发光,24h后恢复,寻常型天疱疮抗体着色无明显变化;免疫印迹检测显示ETA作用后落叶型天疱疮抗原被降解,寻常型天疱疮抗原未受影响。人角质形成细胞与ETA作用后IL-1α分泌受抑制,IL-6的分泌量低于检测水平;ETA作用后细胞上清液分解酪蛋白的活性24h内逐渐增加,36h后活性下降。结论ETA的作用位点在落叶型天疱疮抗原,对寻常型天疱疮抗原无影响;ETA的这种作用具有可逆性,可能通过蛋白酶解作用降解落叶型天疱疮抗原。
Objective To study the action and mechanism of staphylococcal exfoliative toxin A(E-TA)on pemphigus foliaceus antigen(PFA)and pemphigus vulgaris antigen(PVA)expressed on cultured human keratinocytes.Methods Stratified human keratinocytes were incubated with ETA and then stained with sera from patients with pemphigus foliaceus or pemphigus vulgaris as the first antibodies and FITC-la-beled sheep anti-human IgG as the second antibody.Total protein was harvested from the cells pretreated with ETA and run on SDS-PAGE for Western blot with the same antibodies.Simultaneously,supernatants of the keratinocytes before and after ETA treatment were collected for detection of the levels of IL-1α,IL-6with ELISA kits.The caseinolytic activities of the supernatants were tested by spectrometry in which casein was used as a non-specific substrate.Results Down-expression of PFA was shown after ETA treatment while no change of PVA expression was found.The high intensity and continuous linear appearance of fluo-rescent staining before ETA treatment became weak and discontinuous after ETA treatment,which were re-covered gradually in24hours.The degradation of proteins recognized by PF sera after ETV treatment was revealed by Western blot.The decreasing tendency of IL-1αconcentration was found in the supernatants of cell culture after ETA treatment,but IL-6level was too low to be detected.Increased caseinolytic activities were found in the supernatants,and declined36hours after ETA treatment.Conclusions ETA acts on PFA expressed on keratinocytes in vitro,which is reversible along with withdrawal of ETA.The mechanism of E-TA act on PFA may be related to proteolytic action instead of promoting cytokine secretion.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2003年第9期510-512,共3页
Chinese Journal of Dermatology
关键词
天疱疮
表皮剥脱毒素A
抗原
活性
免疫荧光染色
Pemphigus
Exfoliative toxins
Keratinocytes
Interleukines
Peptide peptidohydro-lases
